Tumor antigen

ABSTRACT

Tumor antigen inducing and/or activating HLA-A2-restricted tumor-specific cytotoxic T lymphocytes that is activated by recognizing HLA-A2 and a tumor antigen peptide, and a peptide or polypeptide derived from the tumor antigen, a polynucleotide encoding the peptide or a complementary strand polynucleotide thereof, a transformant comprising a recombinant vector which comprises the polynucleotide are provided.

FIELD OF THE INVENTION

The present invention relates to a tumor antigen, and more particularlyrelates to a peptide or a polypeptide recognized by tumor-specificcytotoxic T lymphocytes, a polynucleotide encoding the peptide or thepolypeptide and a complementary strand polynucleotide thereto, arecombinant vector comprising the polynucleotide, a transformantcomprising the recombinant vector, an antibody against the peptide orthe polypeptide, a compound having any interaction with the peptide orthe polypeptide or the polynucleotide, a cytotoxic T lymphocyte inducerconsisting of the peptide and/or the polypeptide, and a pharmaceuticalcomposition comprising the same, and a method for producing thepolypeptide, a method for screening for a compound having anyinteraction with the peptide or the polypeptide or the polynucleotide, amethod for inducing cytotoxic T lymphocytes using the peptide or thepolypeptide, a method for measuring the peptide or the polypeptide orthe polynucleotide encoding the polypeptide, and a reagent kit used forthe measuring method.

BACKGROUND OF THE INVENTION

The immune system, particularly cytotoxic T lymphocytes (which,hereinafter, may be abbreviated to CTLs) play an important role in theexclusion of cancer in vivo. Infiltration of cytotoxic T lymphocytesexhibiting a cytotoxic activity against tumor cells has been detected atthe tumor site of a cancer patient (Arch. Surg., 126:200-205, 1990.) Atarget molecule (tumor antigen) of the tumor-specific cytotoxic Tlymphocytes was first discovered in a melanoma. A tumor antigengenerated in a tumor cell is degraded in the cell into a peptide (tumorantigen peptide) consisting of 8 to 11 amino acids, which binds to ahuman leukocyte antigen (HLA) molecule that is the majorhistocompatibility complex antigen to be displayed on the surface of thetumor cell. The cytotoxic T lymphocytes recognize a complex consistingof HLA and the tumor antigen peptide, and damage the tumor cell. Inother words, the cytotoxic T lymphocytes recognize the tumor antigenpeptide in an HLA-restricted manner.

HLA is a cell membrane antigen, and is expressed on almost alleukaryotic cells. HLA is mainly classified into class I antigen andclass II antigen. The HLA recognized by the cytotoxic T lymphocytestogether with an antigen peptide belongs to class I antigens. HLA classI antigens are further classified into HLA-A, HLA-B, HLA-C, and so on.It was reported that HLA has genetic polymorphism. The HLA-A2 allele,which is one of polymorphisms of HLA-A subregion, is found inapproximately 23% of African Blacks, approximately 53% of Chinese,approximately 40% of Japanese, approximately 49% of Northern Caucasians,and approximately 38% of Southern Caucasians.

As used herein, a tumor antigen means a protein, a polypeptide, or apeptide, which constitutes part of the tumor cell and is capable ofinducing tumor-specific cytotoxic T lymphocytes. A tumor antigen peptidemeans a peptide that is generated as a result of degradation of thetumor antigen in a tumor cell and can induce or activate tumor-specificcytotoxic T lymphocytes upon being expressed on the cell surface bybinding to an HLA molecule. In addition, the site of the amino acidsequence which is capable of inducing tumor-specific cytotoxic Tlymphocytes that is present in a tumor antigen is called a tumor antigenepitope (tumor antigen determinant.)

In recent years, many genes encoding tumor antigens that can berecognized by cytotoxic T lymphocytes have been identified from cDNA ofhuman tumor cells (Science 254:1643-1647, 1991; J. Exp. Med.183:1185-1192, 1996; J. Immunol. 163:4994-5004, 1999.) Some of thesegenes are involved in cellular proliferation and malignanttransformation, including HER/neu (Proc. Natl Acad. Sci. USA,92:432-436, 1995) mutant cdk (Science, 269:1281-1284, 1995) mutantCASP-8 (J. Exp. Med., 186:785-793, 1997) and so on.

On the other hand, a molecule such as a tumor rejection antigen gene anda T cell antigen receptor (TCR), which are involved in specificimmunity, have been identified in melanoma, esophageal cancer, and othercancers in the past 10 years, and a specific immunotherapy of advancedcancer or metastatic cancer has been studied using the peptide.

Now, in Europe and in the United States, cancer vaccine therapy has beendeveloped in which cytotoxic T lymphocytes are activated by anadministration of a tumor antigen in a cancer patient. Results from aclinical test of a melanoma specific tumor antigen have been reported.For example, administration of a melanoma antigen gp-100 peptidesubcutaneously to melanoma patients along with administeringinterleukin-2(IL-2) intravenously gave a tumor regression in 42% of thepatients (Nature Medicine, 4:321, 1998.) In this way, by utilizing atumor antigen as a vaccine, an effective treatment against cancer can beachieved.

However, almost all of the identified tumor antigens are derived frommelanomas. Tumor antigens derived from epithelial cancers andadenocarcinomas, such as pancreatic cancer, which occur at highincidence rates, have been reported for such specific immunotherapy onlyin a few papers. Pancreatic cancer is one of the largest causes of deathby cancer in the world and causes about 27,000 deaths a year in the USAand about 50,000 deaths in Europe. The main factors causing these largenumbers of deaths are lack of an effective therapeutic method, thedifficulty of diagnosis, and the activity of this cancer. Only 1 to 4%of pancreatic cancer patients have overcome the disease, and theincidence substantially equals the death rate. Therefore, a new approachof therapy, for example, development of specific immunotherapy isneeded.

In addition, in view of the diversity of cancer, an identical tumorantigen should not be expressed in the same degree in all cancer cellsNaturally, cancer vaccine therapy by activating the cytotoxic Tlymphocytes using one kind of tumor antigen has a therapeutic effect oncancer having the tumor antigen. However, in order to induce andactivate the tumor antigen-specific cytotoxic T lymphocytes and obtain ahigh therapeutic effect corresponding to the diversity of cancer, it isimportant to discover and use many novel tumor antigens in accordancewith the diversity of cancer.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a peptide consisting of anamino acid sequence according to any one of those from SEQ ID NO: 1 toSEQ ID NO: 44 is provided.

In another embodiment of the present invention, a polypeptide consistingof an amino acid sequence according to any one of those from SEQ ID NO:45 to SEQ ID NO: 53 is provided.

In still another embodiment of the present invention, a compositioncomprising one or more of peptides or polypeptides selected from thepeptides, which consist of the amino acid sequence according to any oneof those from SEQ ID NO:l to SEQ ID NO:44, and the polypeptides, whichconsist of an amino acid sequence according to any one of those from SEQID NO:45 to SEQ ID NO:53 is provided.

In an additional embodiment of the present invention, an anti-cancervaccine comprising one or more of peptides or polypeptides selected fromthe peptides, which consist of the amino acid sequence according to anyone of those from SEQ ID NO:1 to SEQ ID NO:44, and the polypeptides,which consist of an amino acid sequence according to any one of thosefrom SEQ ID NO:45 to SEQ ID NO:53 is provided.

In yet another embodiment of the present invention, an anti-cancervaccine comprising one or more of peptides or polypeptides selected fromthe peptides, which consist of the amino acid sequence according to anyone of those from SEQ ID NO:1 to SEQ ID NO:44, and the polypeptides,which consists of an amino acid sequence according to any one of thosefrom SEQ ID NO:45 to SEQ ID NO:53, for use in the treatment ofpancreatic cancer, colon cancer, or stomach cancer is provided.

In another embodiment of the present invention, an inducer of cytotoxicT lymphocytes comprising one or more of peptides or polypeptidesselected from the peptides, which consist of the amino acid sequenceaccording to any one of those from SEQ ID NO:1 to SEQ ID NO: 44, and thepolypeptides, which consist of an amino acid sequence according to anyone of those from SEQ ID NO:45 to SEQ ID NO:53 is provided.

In still another embodiment of the present invention, a method forinducing cytotoxic T lymphocytes using one or more of peptides orpolypeptides selected from the peptides, which consist of the amino acidsequence according to any one of those from SEQ ID NO:1 to SEQ ID NO:44,and the polypeptides, which consist of an amino acid sequence accordingto any one of those from SEQ ID NO:45 to SEQ ID NO:53 is provided.

In an additional embodiment of the present invention, a polynucleotideencoding a peptide or a polypeptide consisting of the amino acidsequence according to any one of those from SEQ ID NO:1 to SEQ ID NO:53,or the complementary strand thereof is provided.

In yet another embodiment of the present invention, a polynucleotideaccording to any one of those from SEQ ID NO:54 to SEQ ID NO:62, or thecomplementary strand thereof is provided.

In another embodiment of the present invention, a polynucleotideaccording to any one of those from SEQ ID NO:54 to SEQ ID NO:62, whereina polypeptide encoded by the polynucleotide induces cytotoxic Tlymphocytes and/or is recognized by cytotoxic T lymphocytes, or thecomplementary strand thereof is provided.

In still another embodiment of the present invention, a polynucleotidethat hybridizes to the polynucleotide or the complementary strandthereof under stringent conditions is provided.

In an additional embodiment of the present invention, a recombinantvector comprising the polynucleotide or the complementary strand thereofor the polynucleotide that hybridizes to the polynucleotide or thecomplementary strand thereof under stringent conditions is provided.

In yet another embodiment of the present invention, a recombinantexpression vector comprising the polynucleotide or the complementarystrand thereof or the polynucleotide that hybridizes to thepolynucleotide or the complementary strand thereof under stringentconditions is provided.

In another embodiment of the present invention, a transformanttransformed with the recombinant vector or the recombinant expressionvector, which comprises the polynucleotide or the complementary strandthereof or the polynucleotide that hybridizes to the polynucleotide orthe complementary strand thereof under stringent conditions is provided.

In still another embodiment of the present invention, a method forproducing the polypeptide, which comprises culturing the transformanttransformed with the recombinant expression vector that comprises thepolynucleotide or the complementary strand thereof or the polynucleotidethat hybridizes to the polynucleotide or the complementary strandthereof under stringent conditions is provided.

In an additional embodiment of the present invention, an antibody thatimmunologically recognizes the peptide or the polypeptide is provided.

In yet another embodiment of the present invention, a method forscreening for a compound that enhances at least recognition of thepeptide or the polypeptide by HLA-A2-restricted cytotoxic T lymphocytes,by interacting with the peptide or the polypeptide and/or HLA-A2 toenhance, and/or the compound that enhances expression of thepolynucleotide or the complementary strand thereof by interacting withthe same, is provided wherein the method uses at least one selected froma group consisting of the peptide, the polypeptide, the polynucleotide,the complementary strand thereof, the recombinant vector, therecombinant expression vector, the transformant, and the antibody.

On another embodiment of the present invention, a compound obtained bythe method for screening a compound that enhances at least recognitionof the peptide or the polypeptide by HLA-A2-restricted cytotoxic Tlymphocytes, by interacting with the peptide or the polypeptide and/orHLA-A2 to enhance, and/or the compound that enhances expression of thepolynucleotide or the complementary strand thereof by interacting withthe same, is provided wherein the method uses at least one selected froma group consisting of the peptide, the polypeptide, the polynucleotide,the complementary strand thereof, the recombinant vector, therecombinant expression vector, the transformant, and the antibody.

In still another embodiment of the present invention, a compound thatenhances recognition of at least one of the peptide or the polypeptideby the HLA-A2-restricted cytotoxic T lymphocytes, or the compound thatenhances the expression of the polynucleotide or the complementarystrand thereof by interacting with the same is provided.

In an additional embodiment of the present invention, a pharmaceuticalcomposition used for cancer treatment, comprising at least one selectedfrom a group consisting of the peptide, the polypeptide, thepolynucleotide, the complementary strand thereof, the recombinantvector, the recombinant expression vector, the transformant, theantibody, and the compound is provided.

In yet another embodiment of the present invention, use of thecomposition, the anti-cancer vaccine, the inducer of the cytotoxic Tlymphocytes, or the pharmaceutical composition for cancer disease isprovided.

In another embodiment of the present invention, a method for measuringquantitatively or qualitatively the peptide or the polypeptide, or thepolynucleotide is provided.

In still another embodiment of the present invention, a reagent kit usedin the method for measuring quantitatively or qualitatively the peptideor the polypeptide, or the polynucleotide, is provided wherein the kitcomprises at least one selected from a group consisting of the peptide,the polypeptide, the polynucleotide or the strain thereof, or theantibody.

In an additional embodiment of the present invention, use of a reagentkit for a test of the cancer disease is provided, wherein the kit isused to measuring quantitatively or qualitatively the peptide or thepolypeptide, or the polynucleotide, comprises at least one selected froma group consisting of the peptide, the polypeptide, the polynucleotideor the complementary strain thereof, or the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that OK-CTLp (HLA-A0207/A3101) lyses tumor cells inan HLA-A2-restricted manner.

FIG. 2 illustrates that recognition of the human pancreaticadenocarcinoma cell line Panc-1 by OK-CTLp and interferon-γ productionas a result thereof is an HLA-A2-restricted event.

FIGS. 3A-3B illustrate that OK-CTLp recognizes COS7 cells, in which eachof cDNA clones 1 to 6 (FIG. 3A) and cDNA clone 7 (FIG. 3B), obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, wascoexpressed with HLA-A2, in an HLA-A2-restricted manner.

FIGS. 4A-4F illustrate that cDNA clones 1 to 6, obtained from the humanpancreatic adenocarcinoma cell line Panc-1, are recognized by OK-CTLp ina dose-dependent manner. FIGS. 4A-4F each show that OK-CTLp recognizesCOS7 cells, in which each of cDNA clones 1 to 6, having high homologywith UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, and 2-5 OAS3,respectively, was coexpressed with HLA-A2, in an HLA-A2-restrictedmanner. The symbol—▪—shows the amount of interferon-γ produced byOK-CTLp, when the HLA-A0207 gene was coexpressed with the each tumorantigen gene in target cells,—⋄—shows the amount of interferon-yproduced by OK-CTLp, when the HLA-A2402 gene was coexpressed with eachtumor antigen gene in the target cells.

FIG. 5 illustrates that OK-CTLP or OK-CTL clone recognizes five peptidesderived from a gene product of the tumor antigen gene 1 that is obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, which hashigh homology with UBE2V.

FIG. 6 illustrates that OK-CTLp or OK-CTL clone recognizes four peptidesderived from a gene product of the tumor antigen gene 2 that is obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, which hashigh homology with HNRPL.

FIG. 7 illustrates that OK-CTLp or OK-CTL clone recognizes four peptidesderived from a gene product of the tumor antigen gene 3 that is obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, which hashigh homology with WHSC2.

FIG. 8 illustrates that OK-CTLp or OK-CTL clone recognizes two peptidesderived from a gene product of the tumor antigen gene 4 that is obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, which hashigh homology with EIF4EBP1.

FIG. 9 illustrates that OK-CTLp or OK-CTL clone recognizes threepeptides derived from a gene product of the tumor antigen gene 5 that isobtained from the human pancreatic adenocarcinoma cell line Panc-1,which has high homology with ppMAPkkk.

FIG. 10 illustrates that OK-CTLp or OK-CTL clone recognizes one peptidederived from a gene product of the tumor antigen gene 6 that is obtainedfrom the human pancreatic adenocarcinoma cell line Panc-1, which hashigh homology with 2-5 OAS3.

FIGS. 11A-11F illustrate representative peptides showing that the tumorantigen peptides, which are derived from products of the tumor antigengenes obtained from the human pancreatic adenocarcinoma cell linePanc-1, are recognized by OK-CTL clone in a dose-dependent manner. FIGS.11A-11F show that OK-CTLP recognizes the peptides which are derived fromgene products of the tumor antigen genes 1 to 6, having high homologywith UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, and 2-5 OAS3,respectively.

FIGS. 12A-12C illustrate that three peptides derived from a gene productof the tumor antigen gene 1 having high homology with UBE2V can induceCTL showing cytotoxicity against HLA-A2⁺ tumor cell, from peripheralblood mononuclear cells of a cancer patient. The symbol—▪—shows thehuman pancreatic adenocarcinoma cell line Panc-1(HLA-A0201/1101,)—♦—shows the human colon adenocarcinoma cell line SW620(HLA-A0201/2402,)—◯—shows the HLA-A2 lung adenocarcinoma cell lineRERF-LC-MS (HLA-A1101/1101,)—□ shows an EBV transformed autologous Bcell (HLA-A0207/3101,) and y shows a PHA blast of autologous T cell(HLA-A0207/3101.) These symbols are also used in the same manner inFIGS. 13 to 17 described below.

FIGS. 13A-13B illustrate that two peptides derived from a gene productof the tumor antigen gene 2 having high homology with HNRPL can induceCTL showing cytotoxicity against HLA-A2⁺ tumor cell, from peripheralblood mononuclear cells of a cancer patient.

FIGS. 14A-14B illustrate that two peptides derived from a gene productof the tumor antigen gene 3 having high homology with WHSC2 can induceCTL showing cytotoxicity against HLA-A2⁺ tumor cell, from peripheralblood mononuclear cells of a cancer patient.

FIGS. 15A-15B illustrate that two peptides derived from a gene productof the tumor antigen gene 4 having high homology with EIF4EBP1 caninduce CTL showing cytotoxicity against HLA-A2⁺ tumor cell, fromperipheral blood mononuclear cells of a cancer patient.

FIG. 16 illustrates that one peptide derived from a gene product of thetumor antigen gene 5 having high homology with ppMAPkkk can induce CTLshowing cytotoxicity against HLA-A2⁺ tumor cell, from peripheral bloodmononuclear cells of a cancer patient.

FIG. 17 illustrates that one peptide derived from a gene product of thetumor antigen gene 6 having high homology with 2-5 OAS3 can induce CTLshowing cytotoxicity against HLA-A2⁺ tumor cell, from peripheral bloodmononuclear cells of a cancer patient.

FIGS. 18A-18E illustrate that tumor antigen peptides can induce CTL,which shows cytotoxic activity in an HLA-A2-restricted manner and in adose-dependent manner, from peripheral blood mononuclear cells of acancer patient. FIGS. 18A-18E each show that peptides derived from geneproducts of the tumor antigen genes 1 to 5, having high homology withUBE2V, HNRPL, WHSC2, EIF4EBP1, and ppMAPkkk, respectively, induces CTLthat recognizes the peptide in a HLA-A2-restricted manner, fromperipheral blood mononuclear cells of a cancer patient. Thesymbol—▪—shows T2 cells in which the tumor antigen peptide was made toexpress—⋄—and shows autologous PHA blastoid T cells.

FIGS. 19A-19B illustrate that gene products of the tumor antigen genesKM-PA-2 (FIG. 19A) and KM-PA-4 (FIG. 19B), which were obtained from thehuman pancreatic adenocarcinoma cell line CFPAC-1, are recognized byOK-CTLp in an HLA-A2-restricted manner.

FIGS. 20A-20B show that peptides derived from the tumor antigen genesKM-PA-2 and KM-PA-4, respectively, which were obtained from the humanpancreatic adenocarcinoma cell line CFPAC-1, can induce CTL fromperipheral blood mononuclear cells (PBMC) of a cancer patient, whichshows cytotoxicity against T2 cells which have been pulsed with thepeptide corresponding to the peptide used for stimulating the PBMC(left-hand of FIGS. 20A-20B,) and an HLA-A2⁺ tumor cell Panc-1(right-hand of FIGS. 20A-20B.) FIGS. 21A-21C show that CTL, which wasinduced from peripheral blood mononuclear cells of a cancer patient bypeptide derived from the tumor antigen gene KM-PA-2 obtained from thehuman pancreatic adenocarcinoma cell line CFPAC-1, lyses tumor cells inan HLA-A2-restricted manner.

DETAILED DESCRIPTION OF THE INVENTION

In order to identify a tumor rejection antigen gene and a tumor antigenencoded by the gene, which can be used for specific imnunotherapy forpancreatic cancer, HLA-A2-restricted tumor-specific cytotoxic Tlymphocytes have been established in the present invention that areactivated by recognizing HLA-A2 and a tumor antigen peptide (hereafter,this cell may be called OK-CTLp) from a colon cancer patient, and genesencoding tumor antigens, which can be recognized by these tumor-specificcytotoxic T lymphocytes (CTL,) have been isolated/identified from a cDNAlibrary of Panc-1 cell that as the human pancreatic adenocarcinoma cellline using the gene expression cloning method. In the present invention,genes have also been identified, which can be recognized by CTL in thesame way as described above, from genes identified as those encoding thetumor antigen by the SEREX (Serological analysis of recombinant cDNAexpression libraries) method (Proc. Natl Acad. Sci. USA, 92:11910-11813,1995.) In addition, based on the tumor antigen encoded by the geneobtained, a peptide having the tumor antigen epitope has been found inthe present invention.

As used herein, a polypeptide means a long chain peptide of arbitrarypeptides comprising two or more amino acids bound to each other by apeptide bond or by a modified peptide bond. For example, a protein isincluded in the definition of polypeptide herein. Moreover, a shortchain peptide sometimes called an oligopeptide or an oligomer is simplycalled a peptide herein.

“Recognize” herein means that a subject distinguishes an object fromothers and cognates it, for example, binds to the object cognized.Particularly, in the present invention, CTL which recognize the tumorcells or the tumor antigen peptides means that CTL binds through a Tcell receptor to the tumor antigen peptides that are presented by HLA.

“Activate” herein means to enhance or to make it work further a thing ora state, which has an activity or an action. Particularly, in thepresent invention, activation of CTL means that CTL recognizes anantigen being presented by HLA to produce IFN-γ or CTL showscytotoxicity against the target cells recognized by CTL.

“Induce” herein means to generate an activity or an action from a thingor a state that are in a phase merely having the activity or the action.Particularly, in the present invention, induction of an antigen-specificCTL means to make CTL, which specifically recognizes a certain antigendifferentiate and/or proliferate in vitro or in vivo. In addition, theinducer of cytotoxic T lymphocytes in the present invention means acomposition which changes the state, where CD8-positive T lymphocytesspecifically recognizing a certain antigen is absent or present in avery low degree, to the state, where the cytotoxic T lymphocytesrecognizing the antigen is present in a very degree.

(Isolation and Identification of the Tumor Antigen Gene, Tumor Antigen,and Tumor Antigen Peptide)

In the present invention, OK-CTLp that was HLA-A2-restricted cytotoxic Tlymphocyte described above was used as an effector cell and tumorantigens capable of activating this cell were isolated and identified byusing the gene expression cloning method. In other words, cDNA of thehuman pancreatic adenocarcinoma cell line Panc-1 and cDNA of HLA-A0207were cotransfected into COS-7 cells, and among those cells in which thetransfected genes were expressed, the cells which enhance IFN-γproduction from OK-CTLp were selected, and hence, the gene encoding thetumor antigen capable of activating CTL was identified. The method willbe presented in more detail in examples described herein. As a result,seven cDNA clones were obtained which were recognized by OK-CTLp in anHLA-A2-restricted manner.

In addition, two genes, KM-PA-2 and KM-PA-4, encoding tumor antigenscapable of activating CTL in an HLA-A2-restricted manner were found inthe same manner as described above from the genes encoding the tumorantigens, were detected from a cDNA library of the human pancreaticadenocarcinoma cell line CFPAC-1 using the SEREX method and alreadyreported (Biochim. Biophys. Res. Commun., 281:936-944, 2001,). The SEREXmethod is a method that can be applied to detection of a tumor antigen.However, among 1500 or more kinds of tumor antigens detected by thismethod, those being identified as tumor antigens capable of inducingboth cell-mediated immunity and humoral immunity are only MAGE-1,tyrosinase, and NY-ESO-1. Thus, the tumor antigen identified by theSEREX method cannot always activate CTL. The present invention firstrevealed that the tumor antigen genes described above, KM-PA-2 andKM-PA-4, can activate CTL in an HLA-A2-restricted manner.

All seven cDNA clones described above, which were obtained from Panc-1cell, contained a complete open reading frame (ORF). The nucleotide basesequence of these genes was determined by the Sanger method (ChaihTerminator method) to estimate the amino acid sequence on the basis ofthe nucleotide base sequence. When a homology search was carried out forthese nucleotide base sequences and deduced amino acid sequences in anexisting database such as GenBank, it was found that these genes werecDNAs whose nucleotide base sequences are novel, though they have highhomology with genes as described below. There is no report on the knownhomologous genes functioning as a tumor antigen. With regard to clone 3among the 7 cDNA clones (clones 1 to 7) obtained, the sequence of aninitial clone, which was obtained by the gene expression cloning methoddescribed above, and has high homology with that of a gene ofWolf-Hirschhorn syndrome candidate 2 protein (WHSC2), was 25 bp shorterat the 5′-terminal region than that of WHSC2, so that full-length cDNAwas obtained from the cDNA library of the Panc-1 cell by a standardcolony hybridization method using the clone labeled with ³²p as a probe.Hereinafter, the genes of the Panc-1 cell, from which theabove-described clones 1 to 7 were derived, will be called gene 1 to 7,respectively. Also, the polypeptides consisting of an amino acidsequence encoded by each gene are occasionally herein called geneproduct 1 to 7. Deduced amino acid sequences encoded by each of thesegenes are shown as SEQ ID NO:45 to SEQ ID NO:51, respectively, and thenucleotide base sequences thereof are shown as SEQ ID NO:54 to SEQ IDNO:60, respectively. The above described genes 1 to 6 and the gene 7were registered at the DNA Data Bank of Japan (DDBJ) of the NationalResearch Institute of Genetics in Jun. 12th, 2000 and Aug. 2nd, 2000,respectively (Table 1).

For KM-PA-2 and KM-PA-4, the homologous genes have been reported asshown in Table 1 (Biochem. Biophys. Res. Commun., 281:936-944, 2001.)The nucleotide base sequences of KM-PA-2 and KM-PA-4 are shown as SEQ IDNO: 52 and SEQ ID NO: 53, respectively, and the deduced amino acidsequences are shown as SEQ ID NO:61 and SEQ ID NO: 62, respectively.TABLE 1 CDNA clone (gene) Polypeptide encoded by cDNA Homologous geneBase length (bp) Amino [GenBank [DDBJ SEQ acid SEQ accession accessionno.] ID: NO length ID: NO no.] 1 1775 54 PP 1 270 45ubiquitin-conjugated enzyme [AB044550] variant Kua (UBE2V) [AF155120] 22097 55 PP 2 589 46 heterogeneous nuclear [AB044547] ribonucleoprotein L(HNRPL) [NM_001533] 3 2243 56 PP 3 549 47 Wolf-Hirschhorn Syndrome[AB044549] candidate 2 protein (WHSC2) [AK001304] 4 831 57 PP 4 118 48eIF-4E-binding protein 1 [AB044548] (EIF4EBP1) [NM_004095] 5 2404 58 PP5 779 49 partial putatibe mitogen [AB044546] actibated protein kinasekinase kinase (ppMAPkkk) [AJ242724] 6 6707 59 PP 6 1087 502′,5′-oligoadenylate [AB044545] synthetase 3 (2-5 OAS3) [NM_006187] 7769 60 PP 7 216 51 clevage and polyadenylation [AB046744] specificityfactor (CPSF) [U37012] KM-PA-2 2060 61 PP-KM-PA-2 634 52 KIAA0124 gene[D50914] [AB060694] human homologue of mouse block of proliferatin1(Bop1) [BC005160] KM-PA-4 1841 62 PP-KM-PA-4 142 53 coactosin-likeprotein (CLP) [L54057]

The nucleotide base sequence of gene 1 has high homology with that ofthe ubiquitin-conjugated enzyme variant Kua gene (UBE 2V) registered atGenBank (accession no.AF155120.) The length of the deduced amino acid(aa) encoded by gene 1 was slightly longer than that encoded by theUBE2V gene (3 aa in positions 109 to 111.) The function of UBE2V has notso far been reported.

The nucleotide base sequence of gene 2 has high homology with that ofthe heterogeneous nuclear ribonucleoprotein L (HNRPL) gene (accessionno.NM_(—)001533.) However, the deduced amino acid length was slightlylonger than that of the HNRPL gene at the N-terminal positions 1 to 31.The HNRPL gene product is a heterogeneous nuclear ribonucleoproteincomplex and provides a substrate for the processing events that the mRNAprecursor undergoes in the cytoplasm.

The nucleotide base sequence of gene 3 has high homology with that ofthe Wolf-Hirschhorn syndrome candidate 2 proteins (WHSC2) generegistered at GenBank (accession no.AK001304.) The WHSC2 gene seems tohave some functions in the phenotype of WHS, a multiple malformationsyndrome characterized by mental and developmental defects resultingfrom a partial deletion of the short arm of chromosome 4.

The nucleotide base sequence of gene 4 has high homology with that ofthe eIF-4E-binding protein 1 gene (EIF4EBP1, accession no.NM_(—)004095.)The product of EIF4EBP1 gene is known as a translation initiation factorthat initiates insulin-dependent phosphorylation of 4E-BP1, making itavailable to form an active cap-binding complex.

The nucleotide base sequence of gene 5 has homology with that of thepartial putative mitogen-activated protein kinase kinase kinase gene(ppMAPkkk, accession no.AJ242724) registered at GenBank. The deducedamino acid sequence encoded by gene 5 is 230 aa longer at the N-terminaland 258 aa longer at the C-terminal as compared with that of theregistered ppMAPkkk gene (its function has not yet been reported.)

The nucleotide base sequence of gene 6 consists of 6707 bp and hashomology with that of the 2′,5′-oligoadenylate synthetase 3 gene (2-5OAS3 gene, accession no.NM_(—)006187) with a total of 13-aa differencesat positions 18, 159, 249, 287, 288, 316, 393 to 398, and 984. 2-5 OAS3is known as an IFN-induced protein that plays an important role inimmune-protection against microorganism infection.

The nucleotide base sequence of gene 7 has homology with positions 3701to 4463 of the Human cleavage and polyadenylation specificity factor(CPSF, accession no.U37012) gene and the amino acid sequence isapproximately 1226 aa shorter.

The nucleotide base sequence of gene KM-PA-2 has high homology with thatof the KIAA0124 and human homologues of mouse block of proliferation 1(Bop1.) The nucleotide base at position 1466 of the KIAA0124 gene isguanine (G) and thus, the amino acid residue of the 465^(th) position ishistidine (H). On the contrary, in gene KM-PA-2, they are adenine (A)and arginine (R), respectively.

The nucleotide base sequence of gene KM-PA-4 and the amino acid sequenceencoded by the gene are identical to those of coactosin-like protein(CLP.) In order to obtain tumor antigen peptides from theabove-described 9 genes encoding a tumor antigen, peptides were designedand synthesized based on the amino acid sequences encoded by theabove-described genes. For gene 7, the gene was regarded as a part of agene consisting of a longer base sequence, so that the peptides, whichwere derived from the amino acid sequence encoded by the gene (CPSF)homologous to gene 7, were also designed and synthesized. It has beenknown that a tumor antigen peptide capable of binding to the HLA-A2 hasa motif (a specific sequence) in its sequence. Then, at first, thepeptide having an HLA-A2 binding motif (a specific sequence) wassearched for in the literature (J. Immunol., 152:163, 1994;Immunogenetics, 41:178, 1994,) and the peptides of 9-mer to 11-mer,which were different from each other and suited to the motif obtained,were designed and synthesized based on the amino acid sequence encodedby the above described genes. Recognition of each peptide by CTL wasmeasured using OK-CTLp or several kinds of OK-CTL clones obtained bycloning OK-CTLp by the limiting dilution method using IFN-7 productionfrom these CTL as an indicator. The OK-CTL clones are the cellsrecognizing any one of the above-described genes 1 to 7. On the otherhand, OK-CTLp recognizes any of genes 1 to 7. The results revealed thatOK-CTLp is a cell population recognizing various kinds of tumorantigens. Therefore, when the OK-CTL clones were used to test thepeptide for its ability to activate CTL, a clone was used whichrecognizes the product of the gene encoding the same peptide as that tobe tested. Forty four peptides (SEQ ID NO:1 to SEQ ID NO:44; Table 2 andTable 3) among those synthesized were recognized by OK-CTLp or OK-CTLclones and enhanced IFN-γ production from CTL. Among these peptides, P1to P5, P6 to P9, P10 to P13, P14 and P15, P16 to P18, and P19 are thepeptides consisting of a partial sequence of the amino acid sequencesencoded by gene 1, gene 2, gene 3, gene 4, gene 5, and gene 6,respectively, and encoded also by genes UBE2V, HNRPL, WHSC2, EIF4-EBP1,ppMAPkkk, and 2-5 OAS3 having high homology with each genes. On theother hand, P25, P26, P27, P28, P390, and P31 are the peptidesconsisting of a partial sequence of the amino acid sequences encoded bygene 7 and gene CPSF having high homology with gene 7. P20, P21, P22,P23, P24, P29, and P32 consist of a partial sequence of the amino acidsequence specific to CPSF. TABLE 2 Position SEQ ID NO: of amino inOrigin of acid Amino acid squence Number peptide sequence sequencelisting P 1 Gene 1 43-51 RIQEWCSVI 1 (UBE2V) P 2 64-73 LLLLARWEDT 2 P 385-93 LIADETSGL 3 P 4 201-209 LLQDWHVIL 4 P 5 208-216 ILPRKHHRI 5 P 6Gene 2 140-148 ALVEFEDVL 6 (HNRPL) P 7 404-412 CLYGNVEKV 7 P 8 443-451FMFGQKLNV 8 P 9 501-510 NVLHFFNAPL 9 P10 Gene 3 103-111 ASLDSDPWV 10(WHSC2) P11 141-149 ILGELREKV 11 P12 157-165 MLPLECQYL 12 P13 267-275TLLRKERGV 13 P14 Gene 4 51-59 RIIYDRKFL 14 (EIF4EBP1) P15 52-60IIYDRKFLM 15 P16 Gene 5 290-298 QILKGLLFL 16 (ppMAPkkk) P17 294-302GLLFLHTRT 17 P18 432-440 DLLSHAFFA 18 P19 Gene 6 666-674 QQLCVYWTV 19(2-5 OAS3) P20 CPSF 285-293 SLLYLNQSV 20 P21 CPSF 250-258 KVHPVIWSL 21P22 CPSF 534-542 DMWTVIAPV 22 P23 CPSF 882-890 QLGQGNLKV 23 P24 CPSF392-400 LLLKYTEKL 24 P25 Gene 7 1367-1375 TMLPHHAGL 25 P26 Gene 71296-1304 LLRRADFHV 26 P27 Gene 7 1401-1409 ELLNRYLYL 27 P28 Gene 71358-1366 LIMLQNALT 28 P29 CPSF 797-805 YQLPDWRLV 29 P30 Gene 71359-1368 LMLQNALTIM 30 P31 Gene 7 1358-1367 LIMLQNALTT 31 P32 CPSF456-465 TQLATYSFEV 32

TABLE 3 Position SEQ ID NO: of amino in Origin of acid Amino acidsquence Number peptide sequence sequence listing P 33 KM-PA-2 29-38LEWYDDFPHV 33 P 34 115-124 FSGDVMIHPV 34 P 35 172-180 WAQEDPNAV 35 P 36179-188 AVLGRHKMHV 36 P 37 326-335 RLWEVATARC 37 P 38 348-356 VAWNPSPAV38 P 39 585-593 DLLQNPLLV 39 P 40 586-595 LLQNPLLVPV 40 P 41 612-621VIFHPTQPWV 41 P 42 KM-PA-4 15-24 NLVRDDGSAV 42 P 43 57-65 RLFAFVRFT 43 P44 104-113 VVQNFAKEFV 44

These tumor antigen peptides are-presented on a cell surface by HLA-A2and recognized by T cell receptor (TCR) expressed on thepeptide-specific OK-CTL clone. The peptides derived from gene landUBE2V, gene 2 and HNRPL, or gene 6 and 2-5 OAS3 are recognized by OK-CTLclone expressing TCR-Vβ 8.1, TCR-Vβ 3.2, or TCR-Vβ 14. In addition,Peptides derived from gene 3 and WHSC2, gene 4 and EIF4EBP1, or gene 5and ppMAPkkk are recognized by OK-CTL clone expressing TCR-Vβ13,TCR-Vβ8.1, or TCR-Vβ18, respectively.

Two each of the OK-CTL clones that recognize peptides derived from gene1 and UBE2V, gene 2 and HNRPL, and gene 6 and 2-5 OAS3 were expressedTCR possessing different complementarity-determining regions 3 (CDR 3;an element responsible for binding to antigenic epitopes on a groove ofthe HLA Class I molecules,) respectively. The OK-CTL clone recognizingpeptides derived from gene 3 and WHSC2, gene 4 and EIF4EBP1, and gene 5and ppMAPkkk also expressed TCR possessing the different CDR3,respectively.

Moreover, the above-described 44 peptides recognized by OK-CTLp eachinduced HLA-A2-restricted tumor-specific CTL in vitro from theperipheral blood mononuclear cells (which, hereinafter, may beabbreviated to PBMC) which are autologous cells derived from a coloncancer patient from whom OK-CTLp was obtained, and/or from the PBMC ofHLA-A0201⁺ cancer patients (pancreatic cancer, colon cancer, and stomachcancer.) The above-described CTL induced from the peripheral bloodmononuclear cells of the cancer patients lysed HLA-A2⁺ tumor cells in adose-dependent manner. However, nolysis was observed in the HLA-A2⁻tumor cell RERF-LC-MS, and HLA-A⁺ autologous EBV-B cells and HLA-A⁺ Tcells stimulated by PHA, both of which are derived from normal cells. Inaddition, the above-described CTL showed cytotoxicity in adose-dependent manner against the T2 cells that were pulsed with thesame peptide as that used for stimulation.

On the basis of the examination described above, 44 tumor antigenpeptides capable of activating OK-CTLp were obtained. Furthermore, itwas found that these peptides could induce HLA-A2-restrictedtumor-specific CTLs from PBMC derived from patients of pancreaticcancer, colon cancer, and/or stomach cancer. In addition, it wasrevealed that both the pancreatic adenocarcinoma cell line Panc-1 andthe colon adenocarcinoma cell line SW620 are recognized by CTLp and byCTLs induced from PBMC by the above described peptide. This resultsuggests that pancreatic cancer and colon cancer have a common tumorantigen epitope recognized by host CTLs.

(Polypeptide and Peptide)

A polypeptide according to the present invention is a polypeptideconsisting of the amino acid sequence each of which is encoded by theabove described genes 1 to 7 obtained from the human pancreaticadenocarcinoma cell line Panc-1 or gene KM-PA-2 or KM-PA-4 obtained fromthe human pancreatic adenocarcinoma cell line CFPAC-1, and preferably, apolypeptide consisting of the amino acid sequence described in any oneof those from SEQ ID NO:45 to SEQ ID NO:53. These polypeptides can beused for inducing and/or activating CTL as a tumor antigen. Moreover,these polypeptides can be used as a material for specifying a tumorantigen epitope to obtain a tumor antigen peptide.

A peptide according to the present invention can be obtained bydesigning peptides, for example, which are suited for theHLA-A2-restricted motif, based on the amino acid sequence of theabove-described polypeptide, to select ones recognized by CTL, forexample, which activate and/or induce CTL, from the designed peptides. Apeptide according to the present invention may be the peptide having aproperty of a tumor antigen epitope presented on the surface of anantigen-presenting cell through binding to HLA-A2 and recognized by CTL.The peptide consists of amino acid residues of at least about 5 or more,preferably about 7 or more, and more preferably 9 to 10. Particularlypreferably, the peptide is one consisting of an amino acid sequencedescribed in any one of those from SEQ ID NO:1 to SEQ ID NO:44. Thesepeptides can be used as a tumor antigen peptide for activating and/orinducing the HLA-A2-restricted tumor-specific cytotoxic T lymphocytes.

For activating and/or inducing CTL, one of the above-describedpolypeptide or peptide may be used or they may be used in combination oftwo or more. As described above, CTL is a population consisting ofplural cells that recognize various antigens, so that it is recommendedto use these peptides preferably in combination of two or more.

A polypeptide or peptide, which has one or several amino acid(s) with amutation such as deletion, substitution, addition, or insertionintroduced into the polypeptide or peptide specified as above and isrecognized by at least the HLA-A2-restricted CTL, is also includedwithin the scope of the present invention. A means for introducingmutations such as a deletion, substitution, addition, or insertion iswell known and, for example, Ulmer's technique (Science, 219:666, 1983)can be employed. When introducing such mutation,in view of preventing achange of the fundamental properties (such as the physical properties,activity, or immunological activity) of the peptide, mutual substitutionamong, for example, amino acids having similar properties (polar aminoacids, non-polar amino acids, hydrophobic amino acids, hydrophilic aminoacids, positively charged amino acids, negatively charged amino acids,aromatic amino acids, and so on) can be carried out. In addition, somemodification can be made on these peptides to such an extent that thereis no notable change of their function, such as modification of theconstitutive amino group or carboxyl group.

(Polynucleotide)

A polynucleotide according to the present invention is a polynucleotideconsisting of the nucleotide base sequence described in any one of thosefrom SEQ ID NO:54 to SEQ ID NO:62, which are the nucleotide basesequences of genes 1 to 7 or gene KM-PA-2 or KM-PA-4 obtained from thehuman pancreatic adenocarcinoma cell line Panc-1 or the human pancreaticadenocarcinoma cell line CFPAC-1 as described above, or thecomplementary strand thereof. The polynucleotide may also be apolynucleotide encoding each of the peptides consisting of the aminoacid sequences described in any one of those from SEQ ID NO:1 to SEQ IDNO:44 or a polypeptide consisting of the amino acid sequence describedin any one of SEQ ID NO:45 to SEQ ID NO:53, or the complementary strandthereof. Moreover, the above-described polynucleotide may consist of anucleotide base sequence of at least about 15 or more, preferably about21 to 30 or more nucleotides, wherein the nucleotide base sequencecorresponds to a region encoding a tumor antigen epitope in the aminoacid sequence of the polypeptides according to the present invention, orthe complementary strand thereof. Selection of a useful polynucleotideand determination of its nucleotide base sequence are possible, forexample, by employing well-known protein expression systems to confirmthe ability of the expressed protein to induce and/or activate CTL.

Moreover, a polynucleotide that hybridizes to the above-describedpolynucleotide under stringent conditions is included in the scope ofthe present invention. In the case where the polynucleotide molecule isa DNA molecule, “a DNA molecule that hybridizes to a DNA molecule understringent conditions” can be obtained, for example, by the methoddescribed in “Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, 1989.)” “To hybridize under stringent conditions” hereinmeans that a positive hybridizing signal is still observed even underthe condition in which, for example, hybridization is carried out in asolution containing 6×SSC, 0.5% SDS, and 50% formamide at 42° C. andthen, washing is carried out in a solution containing 0.1×SSC and 0.5%SDS at 68° C.

The above-described polynucleotide can induce and/or activate theHLA-A2-restricted CTL, when it is expressed in cells having HLA-A2. Inthis case, the above-described polynucleotide has a poly(A) structure inits 3′-terminal. The number of poly(A) does not have an influence on thesite encoding the amino acid acting as a tumor antigen, so that thenumber of poly(A,) of the polynucleotide is not limited.

All of the above-described polynucleotides provide genetic informationuseful for producing a polypeptide or a peptide according to the presentinvention or can be also utilized as a reagent and a standard of anucleic acid.

(Recombinant Vector)

A recombinant vector can be obtained by inserting the above-describedpolynucleotide into an adequate DNA vector. The DNA vector used isproperly selected in accordance with the kind of host and the purpose ofuse. The DNA vector may be a naturally existing one and also may be onethat lacks a part of its DNA other than that necessary for replication.For example, vectors can be exemplified as those derived from achromosome, an episome, and a virus, for example, vectors derived from abacterial plasmid, derived from a bacteriophage, derived from atransposon, derived from an enzyme episome, derived from an insertingelement, and derived from an enzyme chromosome element, for example,vectors derived from a virus such as baculovirus, papovavirus, SV40,vacciniavirus, adenovirus, fowl pox virus, pseudo rabies virus, andretrovirus, and vectors prepared by combination of them, for example,vectors derived from the genetic element of the plasmid and thebacteriophage, for example, a cosmid and a phagemid. Further, anexpression vector and a cloning vector etc. can be used in accordancewith the desired purpose.

The recombinant vector, which comprises the constitutional elements ofthe desired DNA sequence and a DNA sequence possessing informationrelating to replication and regulation, such as a promoter, aribosome-binding site, a terminator, a signal sequence, an enhancer, andso on, can be prepared by combining them using well-known methods. As amethod for inserting the polynucleotide according to the presentinvention into the above-described DNA vector, the well-known methodscan be employed. For example, a method can be used, wherein anappropriate restriction enzyme is chosen for treating a DNA to cleave itat a specific site, and then, the DNA is mixed with the DNA used as avector treated in the same way, followed by ligating with a ligase. Adesired recombinant vector can also be obtained by ligating an adequatelinker to the desired polynucleotide followed by inserting the resultantmolecule into a multi-cloning site of a vector suitable for a purpose.

(Transformant)

The DNA vector in which the above-described polynucleotide has beeninserted can be used to obtain a transformant by transforming awell-known host such as Escherichia coli, yeast, Bacillus subtillis, aninsect cell, or a mammalian cell therewith by well-known methods. In thecase of carrying out the transformation, a more preferable system isexemplified by the method for integrating the gene in the chromosome, inview of achieving stability of the gene. However, an autonomousreplication system using a plasmid can be conveniently used.Introduction of the DNA vector into the host cell can be carried out bystandard methods such that described in “Molecular Cloning: A LaboratoryManual” (ed. by Sambrook et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York, 1989.) Concretely, calcium phosphatetransfection, DEAE-dextran-mediated transfection, microinjection, cationlipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction and infection can be employed.

(Producing Polypeptide or Peptide)

Using an expression vector as a DNA vector for transduction of theabove-described transformant, a polypeptide or a peptide according tothe present invention can be provided. A transformant, transformed witha DNA expression vector comprising the above-described polynucleotide,is cultured under well-known culture conditions suitable for each host.Culturing may be conducted by using indicators, such as a function ofthe polypeptide or a peptide according to the present invention that isexpressed by the transformant, particularly at least the activity toinduce and/or activate CTL, or the peptide or the amount of the peptideproduced in the host or outside of the host. Subculturing or batchculturing may be also carried out using an amount of the transformant inthe culture as an indicator.

A peptide according to the present invention can be produced by ageneral method known in peptide chemistry. For example, “PeptideSynthesis (Maruzen) 1975” and “Peptide Synthesis, Interscience, NewYork, 1996” are exemplified. However, any widely known method can beused.

A polypeptide or peptide according to the present invention can bepurified and collected by a method, such as a gel filtrationchromatography, an ion column chromatography, an affinitychromatography, and the like, in combination, or by fractionation meanson the basis of a difference in solubility using ammonium sulfate,alcohol, and the like, using a CTL-activating ability of the polypeptideor the peptide as an indicator. More preferably used is a method,wherein the polypeptide or the peptides are specifically adsorbed andcollected by using polyclonal antibodies or monoclonal antibodies, whichare prepared against the polypeptide or the peptides based on theinformation of their amino acid sequences.

(Antibody)

An antibody according to the present invention is prepared by using theabove-described polypeptide or peptide as an antigen. An antigen may bethe above-described polypeptide or peptide itself, or its fragment thatis composed of at least 5, more preferably at least 8 to 10 amino acids.In order to prepare the antibody specific to the above-describedpolypeptide or peptide, a region consisting of the amino acid sequenceintrinsic to the above-described polypeptide or peptide is desirablyused. The amino acid sequence is not necessarily homologous to the aminoacid sequence of the polypeptide or the peptide, but is preferably asite exposed to outside of a stereo-structure of the polypeptide or thepeptide. In such a case, it is sufficient that the amino acid sequenceof the exposed site is consecutive in the exposed site, even if it maybe discrete in its primary structure. The antibody is not limited aslong as it binds or recognizes the polypeptide or the peptideimmunologically. The presence or absence of the binding or therecognition can be determined by a well-known antigen-antibody bindingreaction.

In order to produce an antibody, a well-known method for antibodyproduction can be employed. For example, the antibody is obtained byadministration of the polypeptide or peptide according to the presentinvention to an animal in the presence or absence of an adjuvant with orwithout linking such to a carrier so as to induce humoral immunityand/or cell-mediated immunity. Any carrier can be used as long as it isnot harmful to the host. For example, cellulose, a polymerized aminoacid, albumin, and the like are exemplified, but not limited thereto. Asan animal used for immunization, a mouse, rat, rabbit, goat, horse, andso on, is preferably used.

A polyclonal antibody can be obtained from serum of an animal subjectedto the above-described immunological means by a well-known method forcollecting antibodies. A preferable means is exemplified byimmunoaffinity chromatography.

A monoclonal antibody can be produced by collecting antibody-producingcells (for example, a lymphocyte derived from a spleen or a lymph node)from the animal subjected to the above-described immunological means,followed by introducing a well-known transformation means with apermanently proliferating cell (for example, myeloma strain such asP3/X63—Ag8 cells.) For example, the antibody-producing cells are fusedwith the permanent proliferating cells by a well-known method to preparehybridomas. Then, the hybridomas are subjected to cloning, followed byselecting ones producing the antibody that recognizes specifically theabove-described polypeptide or peptide to collect the antibody from aculture solution of the hybridoma.

The polyclonal antibody or the monoclonal antibody thus obtained, whichrecognizes and binds to the above-described polypeptide or peptide, canbe utilized as an antibody for purification, a reagent, a labelingmarker and so on.

(Screening and Compound Obtained by Screening)

The above-described polypeptide or peptide, the polynucleotide encodingthe same and the complementary strand thereof, the cell transformedbased on the information concerning the amino acid sequence andnucleotide base sequence, or the antibody immunologically recognizingthe same provide an effective means for screening a substance capable ofinducing and/or activating CTL, when using them solely or in combinationwith each other. The screening method can be constructed utilizing awell-known screening system. For example, as shown in Examples herein,using a system in which the activation of CTL by the antigen-presentingcells that are pulsed with the tumor antigen peptide, is measured on thebasis of the amount of IFN-γ production from CTL. Addition of a testsubstance to the system allows one to select the substance inducingand/or activating CTL and the substance enhancing the induction and/orthe activation. This system describes one screening method, but thescreening method according to the present invention is not limitedthereto.

A compound obtained by the above-described screening method is also partof the present invention. The compound may be a compound enhancing therecognition of the polypeptide or the peptide by CTL through aninteraction with the polypeptide or the peptide according to the presentinvention, and/or HLA-A2. Further, it may be a compound enhancing theexpression of the polynucleotide according to the present inventionthrough an interaction with the polynucleotide. The compound thusselected can be used in a pharmaceutical composition by selecting oneshaving both biological usefulness and low toxicity.

(Pharmaceutical Composition)

The polypeptide or peptide according to the present invention can beused for activating and/or inducing the HLA-A2-restricted tumor-specificcytotoxic T lymphocytes, as a tumor antigen or a tumor antigen peptide.In other words, the method for inducing CTL, which is characterized inthat the above-described polypeptide or peptide is used, and a inducerof CTL comprising the above-described polypeptide or peptide areincluded in the scope of the present invention.

The polypeptide or the peptide according to the present invention, thepolynucleotide encoding the polypeptide and the complementary strandthereof, the recombinant vector prepared based on the information oftheir amino acid sequences and nucleotide base sequences, the celltransformed with the recombinant vector, or the antibody immunologicallyrecognizing the polypeptide or the peptide, the compound enhancing therecognition of the polypeptide or the peptide by CTL through interactionwith the polypeptide or the peptide, and/or HLA-A2, or the compoundenhancing expression of the polynucleotide through interaction therewithcan used a pharmaceutical composition comprising at least one speciesthereof, when using them solely or in combination with each other.

Concretely, for example, composition consisting of the polypeptide orthe peptide according to the present invention, and the pharmaceuticalcomposition comprising the polypeptide or the peptide according to thepresent invention can be used as a so-called anti-cancer vaccine. Insuch a case, in order to activate the cell-mediated immunity, thepolypeptide or the peptide according to the present invention can beused in the presence or absence of an adjuvant with or without linkingsuch to a carrier. Any carrier can be used as long as it is not harmfulto the human body. For example, cellulose, a polymerized amino acid, oralbumin is exemplified, but the carrier is not limited thereto. A dosageform is properly chosen from those to which the well-known means forpreparing a polypeptide or a peptide are applied. The amount thereof tobe administered depends on a degree of recognition of the peptide byCTL, and is generally 0.01 mg to 100 mg/day/adult human body, preferably0.1 mg to 10 mg/day/adult human body as an amount of active substance.Such an amount is administered once every several days or every severalmonths.

Alternately, an effective action of an anti-cancer vaccine can also beobtained by collecting a mononuclear cell fraction from the peripheralblood of a patient, and culturing the fraction with a peptide accordingto the present invention, followed by returning the mononuclear cellfraction, in which CTL are induced and/or activated, back into the bloodof the patient. Culture conditions, such as the concentration ofmononuclear cells and the concentration of the polypeptide or thepeptide according to the present invention when they are cultured, canbe readily determined. Further, a substance, such as interleukin 2(IL-2) having an ability to induce the growth of lymphocytes may beadded to the culture.

In the case of using the polypeptide or the peptide according to thepresent invention as an anti-cancer vaccine, using even only onepolypeptide or one peptide is effective as an anti-cancer vaccine.However, plural kinds of the above-described polypeptide or peptide canbe used in combination. CTL of the cancer patient is the population ofcells recognizing various tumor antigens, so that using plural kinds ofpolypeptides or peptides as an anti-cancer vaccine may give a highereffect than using only one kind.

The above-described composition, inducer of the cytotoxic T lymphocytes,anti-cancer vaccine and pharmaceutical composition are useful fortreatment of a cancer disease such as pancreatic cancer, colon cancer,or stomach cancer.

In addition, the polynucleotide encoding the polypeptide according tothe present invention and the complementary strand thereof are alsouseful for gene therapy of a cancer disease such as pancreatic cancer,colon cancer, or stomach cancer.

A method in which these polynucleotides are present in a vector anddirectly introduced in vivo, and a method in which cells are collectedfrom a donor followed by introducing polynucleotides present in a vectorin vitro, can be both utilized. Retrovirus, adenovirus, and vacciniavirus exemplify the vectors, and retrovirus-related ones are preferred.Needless to say, these viruses show deficiency for replication. Theamount of administration thereof can depend on the degree of recognitionby CTL of the polypeptide encoded by the polynucleotide. Generally, as aDNA content encoding the tumor antigen peptide according to the presentinvention, the amount ranges from 0.1 pg to 100 mg/day/adult human body,preferably 1 pg to 50 mg/day/adult human body. This amount isadministered once every several days or every several months.

(Measuring Method for Diagnosis and Reagent)

The polypeptide or the peptide according to the present invention, thepolynucleotide encoding the polypeptide and the complementary strandthereof, and the antibody immunologically recognizing the polypeptide orthe peptide can be used independently for a diagnostic marker and areagent etc. The present invention also provides a reagent kitcomprising one or more containers in which one or more species thereofare present. For the preparation thereof, it is sufficient to use awell-known means for their preparation according to each of polypeptideor peptide, polynucleotide, or antibody.

Diagnostic means for a disease related to expression or activity of thepolypeptide or the peptide according to the present invention can becarried out, for example, utilizing the interaction or reactivity withthe polynucleotide encoding the polypeptide, by determining the existingamount of the corresponding nucleic acid molecule, and/or determining adistribution of the polypeptide or the peptide in an individual livingbody, and/or determining a presence of the polypeptide or the peptide,and the existing amount in a sample derived from the individual body. Inother words, measurement is carried out quantitatively or qualitativelyfor the polypeptide or the peptide according to the present invention orthe polynucleotide encoding the same as the diagnostic marker. As amethod for quantitative or qualitative measurement of the polypeptide orthe peptide or the nucleic acid encoding the same, which are present inthe sample, a well-known method can be utilized. Radio immunoassay,competitive binding assay, Western blotting analysis, ELISA, and thelike exemplify such a method. In addition, the nucleic acid molecule canbe detected and quantified at an RNA level by using, for example,amplification, polymerase chain reaction (PCR,) RT-PCR, RNaseprotection, Northern blotting method, and other hybridization methods.

The sample subjected to measurement is exemplified by the cells derivedfrom an individual human body present in for example, blood, urine,saliva, spinal fluid, tissue biopsy, or autopsy material, and the like.The nucleic acid molecule subjected to measurement is obtained from theeach sample described above by a well-known method for nucleic acidpreparation. For the nucleic acid molecule, genomic DNA can be directlyused for detection, or it may be enzymatically amplified by using PCR orany other amplification method before the analysis. RNA or cDNA may besimilarly used. In comparing with a normal genotype, a deletion orinsertion can be detected in accordance with a size change of anamplification product. Hybridizing the amplified DNA with the labeledDNA encoding the above-described polypeptide can identify pointmutations.

Detecting mutation of, reduction of, and increase in the polypeptideaccording to the present invention and the DNA encoding the polypeptideby the above-described measuring method, makes it possible to diagnosediseases, to which the polypeptide is associated, such as pancreaticcancer, colon cancer, or stomach cancer.

EXAMPLES

The present invention will be illustrated more concretely with thefollowing examples, but is not limited thereto.

Example 1

(Establishment of HLA-A2-Restricted CTL)

The HLA-A2-restricted tumor-specific cytotoxic T lymphocyte line wasestablished from tumor infiltrating lymphocytes (TIL) of a colon tumorpatient (HLA-A0207/3101, HLA-B46/51, HLA-Cw1) (Int. J. CANCER,81:459-466, 1999; J. Immunol., 163:4994-5004, 1999.) Specifically, TILobtained from the colon tumor patient was cultured for a long period upto 50 days or longer by adding 100 U/ml of recombinant human interleukin2 (IL-2.) Every 7 days, a portion of TIL activated by IL-2 was collectedand cultured together with various kinds of tumor cells or normal cellsto assay its CTL activity by measuring the produced IFN-γ and bymeasuring ⁵¹Cr released from the cancer cells (J. Immunol.,163:4994-5004, 1999.) IFN-γ was measured by an enzyme-linkedimmunosorbent assay (ELISA). At day 58 after the start of the culture,OK-CTLp, which is one of sublines showing HLA-A2-restrictedtumor-specific CTL activity, was obtained. OK-CTLp obtained is a cellpopulation in which 80% of the cells have a phenotype of CD3⁺CD4⁻CD8⁺and 20% of the cells have a phenotype of CD3⁺CD4⁺CD8⁻. Using OK-CTLp asan effector cell and culturing it together with various kinds of cellsthat are used as a target cell, such as a tumor cell, the cytotoxicityto the target cell and activation of OK-CTLp were measured using the⁵¹Cr-release test and by using IFN-γ production as an indicator,respectively. The results are each presented in FIG. 1 and FIG. 2.

OK-CTLp obtained, as shown in FIGS. 1 and 2, recognized HLA-A0201⁺Panc-1pancreatic adenocarcinoma cell, SW620 colon adenocarcinoma cell,HLA-A0206⁺KE3 esophageal squamous-cell carcinoma (SCC) cell andHLA-A0207⁺CA9-22 oral SCC cell to produce IFN-γ and represent sufficientcytotoxicity. However, no cytotoxicity was shown against HLA-A2⁻ tumorcells, such as QG56 lung adenocarcinoma cell, RERF-LC-MC lungadenocarcinoma cell, and COLO320 colon adenocarcinoma cell, andautologous Epstein-Barr virus transformed B cell (EBV-B) and autologousphytohemagglutinin (PHA)-blastoid T cells both derived from the normalcells. Further, OK-CTLp lysed all of the HLA-A2⁺ tumor cells tested (R27breast cancer cell, HAK-2 primary hepatocellular carcinoma cell,SK-MEL-5 melanoma cell, and SF126 astrocytoma cell, which areHLA-A0201⁺, and HLA-A0206⁺ PC9 lung adenocarcinoma cell, and 1-87 lungadenocarcinoma cell and OMC-4 cervical SCC cell, which are HLA-A0207⁺.)The CTL activity was inhibited by an anti-HLA class I monoclonalantibody (mAb), an anti-CD8 mAb or an anti-HLA-A2 mAb, but not inhibitedby other mAbs (FIG. 2.) This result revealed that OK-CTLp recognizes theabove-described tumor cells in a HLA-A2-restricted manner and showscytotoxicity.

Meanwhile, the genotype of the HLA class I alleles of theabove-described tumor cells has been disclosed (J. Immunol.,163:4994-5004, 1999.) The serotype of the HLA class I of theabove-described patients was determined by applying a conventionalmethod using the peripheral blood mononuclear cells (PBMC). In addition,the HLA-A2 subtype was determined by a sequence-specific oligonucleotideprobe method and direct DNA sequencing. The phenotype of OK-CTLp wasanalyzed by direct immunofluorescence analysis using anti-CD3 mAb,anti-CD4 mAb, or anti-CD8 mAb (made by Nichirei) or anti-TCRaB mAb(WT31, Becton Dickinson), which were labeled with fluoresceinisothiocyanate (FITC). In addition, the antibodies used to analyze forHLA-A2 restriction and specificity of OK-CTLp were anti-HLA class I mAb(W6/32, IgG2a,) anti-HLA-A2 mAb (BB7.2, IgG 2b,) anti-CD8 b (Nu-Ts/c,IgG2a,) anti-HLA class IImAb (H-DR-1, IgG2A,) and anti-CD4imAb (Nu-Th/i,IgG1.) Anti-CD13 mAb (MCS-2, IgG2a) and Anti-CD14 mAb (JML-H14, IgG1)were used as an isotype-matching control mAb.

Example 2 Isolation and Identification of cDNA Clone Encoding TumorAntigen

A gene encoding the tumor antigen of the Panc-1 tumor cell recognized byOK-CTLp was isolated and identified according to the well-known geneexpression cloning method (J. Immunol., 163:4994-5004, 1999.)Specifically, poly(A)⁺RNA of the Panc-1 tumor cells was converted tocDNA, and ligated with a SalI adapter so as to insert into theexpression vector pCMV-SPORT-2 (Invitrogen Corp.)

cDNAs of HLA-A0207, HLA-A2402, or HLA-A2601 were obtained by reversetranscriptase polymerase chain reaction (RT-PCR) and cloned into theeukaryote expression vector pCR3 (Invitrogen Corp.)

200 ng of the above-described plasmid DNA pool or clones of Panc-1 cellcDNA library was mixed with 200 ng of the cDNA of HLA-A0207 in 100 μl ofOpti-MEM (Invitrogen Corp.) for 30 min. 50 μl of this mixture was addedto COS-7 cells (5×10³) and incubated for 6 h in a 96-well U-bottom typemicroculture plate (Nunc Corp.) for co-tranduction. Then, RPMI-1640culture medium containing 10% FCS was added to and culturing was carriedout for 2 days, followed by the addition of OK-CTLp (5×10⁴) to eachwell. After a further 18 h incubation, 100 μl of the supernatant wascollected and IFN-γ production was measured thereon by ELISA. In thiscase, using COS-7 cells to which the gene had not been transfected as atarget, IFN-γ production by OK-CTLp was examined and the value of IFN-γproduced was subtracted as a background from that of each measurement.As a result, seven cDNA clones were obtained, which enhanced IFN-Yproduction by OK-CTLp through recognition by OK-CTLp.

The nucleotide sequence of the seven cDNA clones obtained was determinedby dideoxynucleotide sequencing method using a DNA sequencing kit(Perkin Elmer Corp.) and using an ABI PRISM™ 377DNA sequencer (PerkinElmer Corp.) In addition, the amino acid sequence encoded by each clonewas deduced from the nucleotide base sequence. Also, a homology searchof the nucleotide base sequence of these clones was conducted throughaccessing GenBank. The results are presented in the above-describedTable 1. With regard to clone 3 among the seven cDNA clones (clones 1 to7) obtained, the sequence of an initial clone, which was obtained by thegene expression cloning method described above, was 25 bp shorter at the5′-terminal region than that of WHSC2 showing high homology, so thatfull-length cDNA was obtained from the cDNA library of the Panc-1 cellby a standard colony hybridization method using the clone labeled with³²P as a probe.

As shown in FIGS. 3A and 3B, clones 1 to 7 were each recognized byOK-CTLp to enhance IFN-γ production of OK-CTLp. However, OK-CTLp did notrecognize COS-7 cells to which HLA-A0207 cDNA and the cDNA clone used asa negative control were cotransfected, or COS-7 cells to which any oneof cDNA clones 1 to 7 and the cDNA of HLA-A2402 or HLA-A2601 werecotransfected, and did not show the IFN-γ production. When variousconcentrations of cDNA clones 1 to 6 were cotransfected into COS-7 cellstogether with 100 ng of HLA-A0207 cDNA or HLA-A2402 cDNA, IFN-γproduction by OK-CTL was observed in a dose-dependent manner (FIGS. 4Ato 4F.)

Expression of the mRNA of these genes was examined by Northern blottinganalysis. The same expression pattern was observed except for gene 5.These genes are expressed commonly in the tumor cells and normal cells.However, expression levels in tumor cells such as Panc-1 cell, SW620cell, and CA9-22 cell were significantly higher than that in normalcells, such as the T cell stimulated by PHA and a B cell transformed byEpstein-Barr virus (EBV-B). Expression of mRNA of gene 5 was barelydetected under these experimental conditions. The reason may be thatexpression of gene 5 is rare as proven by the fact that colonyhybridization using clone 5 labeled with ³²P gives only 3 clones from anabout 1×10⁶ library.

Example 3 Establishing OK-CTL Clone

Since CTL activated by recognizing the tumor antigen is a population ofcells recognizing plural kinds of tumor antigens, the above-describedOK-CTLp was subjected to cloning by limiting dilution culture (0.3, 0.5,1, 2, and 4 cells/well) to obtain an OK-CTL clone (J. Immunol.,163:4994-5004, 1999.) These clones are those having CTL activityselected by culturing them together with COS-7 cells into which 100ng/well of any one of the above-described seven cDNA clones and 100ng/well of HLA-A0207 cDNA were cotransfected, or with the tumor cells,in a cell ratio of 1:1, and measuring IFN-γ production. Specifically,three hundred CTL clones were obtained from the parent line OK-CTLp by alimiting dilution culture. Eighty CTL clones among them hadHLA-A2-restricted tumor-specific CTL activity and expressed thephenotype of CD3⁺CD4⁻CD8⁺ and TCR odd+. Among them, 2, 3, 1, 3, 2, and 4CTL clones showed reactivity to the COS-7 cells expressing clone 1,clone2, clone 3, clone 4, clone 5, and clone6, respectively. In otherwords, it was revealed that the tumor antigen recognized by CTL differsin accordance with the CTL clones. Table 4 shows data of fifteen typicalCTL clones. This suggested that OK-CTLp, i.e., CTL derived from thecancer patient, is a population of cells recognizing plural kinds oftumor antigens. TABLE 4 cDNA expressed in COS-7 cell together withHLA-A0207 cDNA clone 6 clone 1 clone 2 clone 3 clone 4 Clone 5 (2-5 noCTL clone (UBE2V) (HNRPL) (WHSC2) (EIF4EBP1) (ppMAPkkk) OAS3) cDNAPanc-1 2-2-H3 110 0 0 0 0 0 0 340 2-1-H12 134 0 0 0 0 0 0 264 1-2-D7  0200  0 0 0 0 0 235 1-2-D12  0 >1000     0 0 0 0 0 >1000 4-1-H8  0 133  00 0 0 0 84 4-2-A11  0 0 100  0 0 0 0 725 0.5-1-H12  0 0 0 >1000     0 00 >1000 0.5-1-D6  19 0 0 118  0 13  0 448 4-2-B3  0 0 0 95  0 0 0 1002-1-F4  0 0 0 0 >1000     0 0 >1000 0.5-1-H2  0 0 0 0 81  0 0 1220.5-1-D7  27 34  0 0 0 110  0 304 0.5-2-A4  0 0 0 0 0 113  0 >10001-2-D1  21 0 22  44  0 61  0 78 2-2-B4  0 0 0 0 0 >1000     0 >1000

Example 4 Preparation of Tumor Antigen Peptide and its CTL-InducingActivity

In order to obtain the tumor antigen peptide derived from the seventumor antigen genes, which were obtained in Example 2 and can induce CTLin a HLA-A2-restricted manner, a peptide having an HLA-A2 binding motif(a specific sequence) was searched for in the literature (J. Immunol.,152:163, 1994; Immunogenetics, 41:178, 1994,) and peptides of 9-mer to11-mer, which were different from each other and suited to the motifobtained, was designed and synthesized based on the amino acid sequenceencoded by the above-described genes 1 to 7 and the amino acid sequenceof UBE2V, HNRPL, WHSC2, EIF4EBP1, ppMAPkkk, 2-5 OAS3, and CPSF havinghigh homology with these genes. The purity of the peptides obtained waseach 70% or higher.

Binding activity of the peptide to the HLA-A0201 molecule was testedusing a T2 cell mutant strain (Cancer Res., 54:1071-1076, 1994.) The T2cell expresses the HLA-A2 molecule on a cell surface without binding toa peptide, because of deficiency of TAP. Specifically, the synthesizedpeptide (10 μM) and the T2 cells were incubated at 26° C. for 3 h and,subsequently, incubated in 5% CO₂and 95% air at 37° C. for 3 h. Thus, T2cells, on whose surface the peptide was presented by HLA-A2, wereobtained. The cells were incubated together with anti-HLA-A2 mAb (BB7.2)followed by staining with R-phycoerythrin linked F (ab′)₂ rabbitanti-mouse immunoglobulin (Ig) (DAKO Corp.) Then, the expression patternwas analyzed by employing FACScan (Beckman Dickinson Corp.), whichresulted in confirmation that HLA-A0201 molecules with the peptide wereexpressed on the cell surface.

In order to test for recognition of a peptide by CTL, the T2 cellspreviously pulsed with each peptide (10 μM) was used as a target cell(T), and OK-CTLp or OK-CTL clone was used as an effector cell (E). Thetarget cell and the effector cell were incubated for 18 h, thesupernatant collected, followed by measuring IFN-γ contained in thesupernatant by an ELISA. In the case where OK-CTLp was used as theeffector, an E/T ratio was set to 10:1. In the case where the OK-CTLclone was used, it was set to 2:1 to conduct the test. On the otherhand, in case where the OK-CTL clone was used to test for the CTLactivating ability of the peptide, the clone was used which recognizedthe gene product encoding the peptide being examined. Using IFN-γproduction of OK-CTLp or OK-CTL clone against the T2 cells, which hadnot been pulsed with the peptide, as a background, subtraction wasperformed from each measurement value. The results were shown in FIGS. 5to 10. FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, and FIG. 10 each show theresult of the peptide derived from gene 1 and UBE2V, gene 2 and HNRPL,gene 3 and WHSC2, gene 4 and EIF4EBP1, and gene 5 and ppMAPkkk, and gene6 and 2-5 OAS3.

Moreover, the various concentrations of each peptide were used forincubation together with the T2 cells to examine the CTLclone-activating ability, resulting in finding that the CTL clone can beactivated by each peptide in a dose-dependent manner. Representativeexamples of peptides derived from gene 1 and UBE2V, gene 2 and HNRPL,gene 3 and WHSC2, gene 4 and EIF4EBP1, gene 5 and ppMAPkkk, and gene 6and 2-5 OAS3 were presented in FIGS. 11A-11F, respectively. On the otherhand, Table 5 shows peptides derived from gene 7 and CPSF. In Table 5,the peptide derived from EBV and the peptide derived from influenzavirus are positive controls, which can activate CTL.

As the result of these experiments, it was revealed that the peptidesshown in Table 2 described-above can activate OK-CTLp and/or OK-CTLclone to produce IFN-γ. TABLE 5 IFN-γ production (pg/ml) Concentrationof peptide Peptide 0.1 μM 1 μM 10 μM 50 μM Derived 0 93 693 35 frominfluenza virus Derived 119 390 371 117 from EBV P20 344 643 0 34 P21428 501 81 195 P22 254 1027 431 407 P23 6360 0 345 160 P24 728 Detection103 2 limit or higher P25 69 1569 25 122 P26 141 418 1239 0 P27 352 889250 0 P28 898 Detection 0 144 limit or higher P29 0 Detection 86 362limit or higher P30 88 7001 Detection Detection limit or higher limit orhigher P31 443 Detection Detection 314 limit or higher limit or higher

Example 5 CTL Induction from Peripheral Blood Mononuclear Cells Derivedfrom Cancer Patient by a Peptide

Among the peptides that were able to enhance IFN-γ production from theOK-CTLp or OK-CTL clone in Example 4, those from P1 to P19 were examinedfor their ability to induce CTL from peripheral blood mononuclear cellsderived from a cancer patient. The method for inducing CTL by a peptidewas according to the well-known method (J. Exp. Med., 187:277-288, 1998;Cancer Res., 59:4056-4063, 1999.) Specifically, autologous peripheralblood mononuclear cells (PBMC) derived from a cancer patient, from whomOK-CTLp was obtained, were incubated together with the peptide (10 μM.)The cells were re-stimulated at day 10 and 14 after the start ofculturing, using autologous PBMCs as an antigen presenting cell (APC)which were pulsed with 10 μM of the same peptide for 2 h and exposed toirradiation (30 gray.) At day 21 after the start of culturing, the cellswere collected to test for surface phenotype. In addition, the cellswere examined for the recognition of various target cells, using IFN-γproduction measured by ELISA when they were cultured together with thetarget cells as an indicator. As a target cells, SW620 cell, CA9-22cell, and Panc-1 cell, which are the HLA-A2⁺ tumor cells, were used. Theresult was shown in Table 6. TABLE 6 IFN-γ production (pg/ml) fromperipheral blood mononuclear cells in recognition of various targetcells QG56 RERF-LC-MS COLO320 SW620 CA9-22 Panc-1 (HLA-A26/ (HLA-A11/(HLA-A24/ (HLA-A0201/ (HLA-A0207/ (HLA-A0201/ Peptide MFI 26) 11) 24)24) 24) 11) P1 571 0 26 50 235 81 492 P2 607 0 0 5 53 0 0 P3 910 0 0 44188 58 289 P4 1008 0 0 0 60 0 0 P5 637 0 0 38 500 96 638 P6 819 0 8 40344 863 527 P7 783 0 0 0 344 0 54 P8 499 0 26 0 142 165 186 P9 832 0 270 194 98 339 P10 504 0 0 0 108 130 163 P11 1089 0 0 0 893 62 >1000 P12780 0 0 40 46 0 197 P13 656 0 15 0 151 95 115 P14 591 0 0 0 112 184 265P15 789 0 32 0 199 219 502 P16 887 0 0 0 0 147 113 P17 660 0 00 >1000 >1000 691 P18 657 29 0 0 >1000 70 >1000 P19 775 0 30 66 55 48105 no 491 0 0 36 0 0 17 Peptide

PBMC stimulated in vitro using P1, P3, P5, P6, P8, P9, P10, P11, P13,P14, P15, P17, or P18 among the 19 peptides recognized SW620 cells,CA9-22 cells and Panc-1 cells, which are the HLA-A2⁺ tumor cells,produced IFN-γ in a significant amount. However, the PBMC barelyrecognized the HLA-A2⁻ tumor cell. On the other hand, P2, P4, P7, P12,and P16 also induced CTL recognizing any one of the HLA-A2⁺ tumor cells.PBMC stimulated by P19 recognized not only the tumor cells expressingHLA-A2, but also the tumor cells expressing other types of HLA. IFN-γproduction from these CTL was inhibited by anti-HLA class I mAb,anti-CD8 mAb, or anti-HLA-A2 mAb, and not inhibited by other mAbs. Inaddition, when peripheral blood mononuclear cells (PBMC) were preparedfrom blood of two HLA-A0201+ patients (a colon cancer patient and apancreatic cancer patient) to examine CTL induction by the peptides, thesame result as above was obtained. In other words, it was found that theabove-described peptides can induce HLA-A2-restricted CTL from theperipheral blood mononuclear cells of the patient.

Binding affinity of the peptides to HLA-A0201 molecule was expressed asa relative mean fluorescence intensity (MFI) of the HLA-A2 molecule. TheMFI of positive and negative controls were 898 and 490, respectively. Itcan be supposed that binding affinity of a peptide to the HLA-A0201molecule has no correlation with induction of CTL by the peptide.

Moreover, CTL-activating ability of these 19 peptides was directlyexamined in a ⁵¹Cr-releasing test using toxicity to the target cells asan indicator. Specifically, the above-described PBMC, in which CTL wasinduced, was further recultured to proliferate in the presence ofautologous APC, IL-2, and a corresponding peptide. At about day 21 to 28after the start of reculturing, PBMC was collected and cytotoxicitythereof was tested again by measuring IFN-γ and by a 6-h ⁵¹Cr-releasingtest. The results were shown in FIGS. 12 to 17. PBMC of the cancerpatient, which was stimulated by these peptides, lysed the HLA-A2⁺ tumorcells. However, the autologous EBV-B cells and the T cells stimulated byPHA, both of which were derived from normal cells and were expressingHLA-A2, and the HLA-A2⁻ tumor cell RERF-LC-MS, were not lysed. However,PBMC stimulated by peptides P14, P15, and P17 also showed cytotoxicityto the autologous EBV-B cells. Moreover, PBMC stimulated by P19 showedhigh cytotoxicity to the autologous EBV-B cells. In addition, PBMCstimulated by these peptides showed cytotoxicity to the T2 cells, whichwas pulsed with the same peptide as that used for stimulation of PBMC,in a dose dependent manner. Typical examples are shown in FIGS. 18A-18E.From these above, it was found that the above-described peptides caninduce CTL, which shows HLA-A2-restricted cytotoxicity, from theperipheral blood mononuclear cells of the cancer patient.

Example 6 CTL Induction from Peripheral Blood Mononuclear Cell ofPatient by Peptide

For six peptides (P21, P22, P24, P26, P30, and P32) with a purity of 95%or higher among peptides from P20 to P32 derived from gene 7 and CPSFamong the tumor antigen peptide obtained in Example 4, the activity toinduce CTL in vitro from human peripheral blood mononuclear cells (PBMC)was examined using IFN-γ production as an indicator. PBMC used wasprepared from each peripheral blood of sixteen HLA-A2-positive cancerpatients (4 patients with pancreatic cancer, seven patients with stomachcancer, and 5 patients with colon cancer) and six healthy individuals.Specifically, 1×10⁵ PBMC was added to each well of 96-well U-bottom typemicroculture plate (Nunc Corp.) and incubated together with 10 μM ofeach of the above-described peptides in 200 μl culture medium. Theculture medium consisted of 45% RPMI-1640, 45% AIM-V (Invitrogen Corp.,)10% fetal calf serum (FCS), 100 U/ml of human interleukin-2, and 0.1 μMMEM nonessential amino acid solution (Invitrogen Corp.). At day 4 andday 7 after the start of culturing, half of the culture medium wasremoved and replaced with the above-described composition containingeach corresponding peptide. At-day 10 after the start of culturing, thecells were collected and washed, followed by reacting with T2 cells,which were pulsed with each corresponding peptide, to measure the amountof IFN-γ produced.

Meanwhile, the above-described cells cultured for 10 days afterstimulation by the peptide were further cultured for 10 days. Thecytotoxicity of the obtained cells against the Panc-1 pancreaticadenocarcinoma cell (HLA-A2) was measured by the standard 6-h⁵¹Cr-releasing test in an E/T ratio of 10:1. The results obtained arepresented as % specific lysis (table 7). Together with this step,cytotoxicity against SSTW-9 tumor cell as the HLA-A2 tumor cell wasmeasured to use as a background that was subtracted from theabove-described result.

As a result, induction of HLA-A2-restricted CTL from PBMC by P21, P22,P23, P26, P30, and P32, which was specific to each peptide, was observedin patients of 31% (5/16,) 38% (6/16,) 25% (4/ 6,) 31% (5/16,) 44%(7/16,) and 7% (1/16) of the above-described sixteen patients,respectively. On the other hand, induction of CTL by P21 and P22 fromthe PBMC of healthy individuals was found in 50% (3/6) and 33% (2/6,)respectively. However, the other peptides did not induce CTL from thePBMC of healthy individuals (Table 7). The above-described CTL inducedfrom PBMC of the cancer patients by using the peptide showedcytotoxicity against Panc-1 pancreatic adenocarcinoma cell, and alsoSW620 colon adenocarcinoma cell (HLA-A2/A24) and KWS stomachadenocarcinoma cell (HLA-A2), both of which are the HLA-A2⁺ tumor cells.However, lysis was not observed in SSTW9 stomach adenocarcinoma cell(HLA-A24), which is the HIA-A2⁻ tumor cell, or the PHA-blastoid T cellsor the EBV-B transformed B cells, both of which express HLA-A2 and arenot the tumor cells. Recognition of the tumor cell by theabove-described CTL was inhibited by anti-HLA class I mAb, anti-CD8 mAb,or anti-HLA-A2 mAb, and not inhibited by other mAb. TABLE 7 INF-γproduction (pg/ml)/% specific lysis Origin of PBMC P21 P22 P23 P26 P30P32 pancreatic 1 0 0 0 0 0 0 cancer 2 7 38/37 41/53 35/36 12 12 patient3 0 0 0 0 0 0 4 543/12  8 72/10 77/20 73/11 0 stomach 5 0 0 0 53/11 0 0cancer 6 0 0 0 0 0 0 patient 7 0 0 46/10 0 0 0 8 5 0 0 11 146/18  0 91500</27    0 0 8 92 0 10 0 0 0 0 46/26 0 11 136/18  0 0 0 0 0coloncancer 12 0 92/28 71/18 0 0 57/18 patient 13 0 179 138/10  0 41/120 14 66/11 80/19 48 0 33/10 0 15 0 5 14 40/12 0 0 16 140 0 0 45 68 4healthy HD1 0 0 0 0 0 0 individuals HD2 0 0 13 0 0 0 HD3 58 0 8 0 14 0HD4 5 0 6 0 0 0 HD5 224 0 83 13 0 0 HD6 97 14 45 0 0 0

Example 7 Isolation and Identification of cDNA Encoding Tumor Antigen

From the genes encoding the tumor antigens (Biochim. Biophys. Res.Commun., 81:936-944, 2001), which were detected by the SEREX(Serological Analysis of Recombinant cDNA Expression Libraries) method(Proc. Natl Acad. Sci. USA, 92:11910-11813, 1995) and already reported,two tumor antigen genes, KM-PA-2 and KM-PA-4, which can activateHLA-A2-restricted CTL, were found by using the same method as that ofExample 2. Specifically, cDNA clones each encoding KM-PA-2, KM-PA-4,KM-PA-5, KM-PA-14, KM-PA-15, or KM-PA-18 were packaged in pBluescriptvector, and digested by EcoRI and XhoI to insert into pCMV-SPORT2. ThesecDNAs in various concentrations were each coexpressed in COS-7 cellstogether with HLA-A0207 or HLA-A2402. Using the COS-7 cells as a targetcell, incubation was performed together with OK-CTLp. As a result, COS-7cells, to which KM-PA-2 or KM-PA-4 was cotransfected with HLA-A0207,induced IFN-γ production from OK-CTLp, in a dose dependent manner of thegene (FIGS. 19A-19B.) The SEREX method is a method for detection of thetumor antigen. However, among 1500 or more kinds of tumor antigensdetected by this method, those identified as tumor antigens capable ofinducing both cell-mediated immunity and humoral immunity are onlyMAGE-1, tyrosinase, and NY-ESO-1. Therefore, even a gene identified bythe SEREX method as encoding the tumor antigen cannot always activateCTL. It was first found that the above-described tumor antigen genes,KM-PA-2 and KM-PA-4, can activate CTL in an HLA-A2-restricted manner.

Example 8 Preparation of Tumor Antigen and Activity for Inducing CTLfrom PBMC of Cancer Patient

In order to obtain the tumor antigen peptide derived from the tumorantigen genes, KM-PA-2 and KM-PA-4, obtained from Example 7, differentpeptides of 9-mer or 10-mer were designed based on the amino acidsequences encoded by KM-PA-2 and KM-PA-4 and synthesized by a well-knownmethod, in the same way as that in Example 4.

The ability of the synthesized peptide to induce CTL from the peripheralblood mononuclear cells of the colon cancer patient, from whom OK-CTLpwas obtained, was examined in the same way as that in Example 6. As aresult, as shown in FIGS. 20A-20B, PBMC that was stimulated in vitrousing any one of peptides P33 to P41 (SEQ ID NO: 33 to SEQ ID NO:41)derived from KM-PA-2 and peptides P42 to P44 (SEQ ID NO:42 to SEQ IDNO:44) derived from KM-PA-4, produced IFN-γ through recognition of theT2 cells (left-hand figure of FIGS. 20A-20B,) which were pulsed with thepeptide corresponding to that used for stimulation of the PBMC, and thePanc-1 cell (right-hand of FIGS. 20A-20B,) which is the HLA-A2⁺ tumorcell. However, the PBMC reacted merely to the HLA-A2⁻ tumor cell. As aresult, it was revealed that any one of the above-described twelvepeptides can induce the antigen specific CTL from PBMC of the cancerpatient in an HLA-A2-restricted manner and that the induced CTL canrecognize the above-described peptides to produce IFN-γ in anHLA-A2-restricted manner. In addition, cytotoxicity of these CTLsinduced by the peptides was directly confirmed by the ⁵° Cr-releasingtest in the same way as that in Example 6. FIGS. 21A-21C show arepresentative example of the result. As shown in FIGS. 21A-21C, CTLsinduced from PBMC of the cancer patient by P35, P39, or P40 lysed Panc-1cells and YPK-3 cells, both of which are HLA-A2⁺ tumor cells. However,the HLA-A2⁻ tumor cell PaCa-2, the EBV-B cell line OKAB2, and the Tcells stimulated by PHA, was not lysed. In other words, it was revealedthat the above-described peptides can induce CTL showing cytotoxicityfrom the peripheral blood mononuclear cells of the cancer patient in anHIA-A2-restricted manner. Further, CTL was also induced by theabove-described peptides from PBMC obtained from the cancer patient,such as a pancreatic cancer patient, in addition to the colon cancerpatient from whom OK-CTLp was obtained.

Example 9

In order to determine the phenotype of TCR expressed on the cell surfaceof the CTL clone recognizing the above-described peptide, total RNA ofeach clone was obtained from each 5×10⁶ CTL clones, which were obtainedin Example 3, using RNAzol™ B (TEL-TEST Corp.) cDNA was prepared usingthe SuperScrip™ Preamplification System (Invitrogen Corp.) for firststrand cDNA synthesis. Single stranded cDNA was amplified by polymerasechain reaction (PCR) using one of the 22 different Vβ primers (Vβ1 to20) and 3′C β primers. PCR was performed for 35 cycles, wherein 1 cyclecomprised denaturation at 94° C. for 1 min, annealing at 58° C. for 2min, and extension at 72° C. for 3 min. The PCR product was insertedinto plasmid pCR2 followed by transformation into Escherichia coli usingthe TA cloning system (Invitrogen Corp.), selection of colonies, andplasmid preparation for determining the cDNA sequence.

As the result, two each of CTL clones reacting with the peptides derivedfrom UBE2V and gene 1, the peptide derived from HNRPL and gene 2, andthe peptide derived from 2-5 OAS3 and gene 6, expressed TCR-Vβ 8.1,TCR-Vβ 3.2, and TCR-Vβ 14, respectively. In addition, CTL clonesrecognizing the peptide derived from WHSC2 and gene 3, the peptidederived from EIF4EBP1 and gene 4, the peptide derived from ppMAPkkk andgene 5, expressed TCR-Vβ 13.1, TCR-Vβ 8.1, and TCR-Vβ 18, respectively(Table 8-1 and Table 8-2).

Two each of CTL clones recognizing the peptide derived from UBE2V andgene 1, the peptide derived from HNRPL and gene 2, and the peptidederived from 2-5 OAS3 and gene 6, expressed TCR possessing differentcomplementarity-determining regions 3 (CDR3) (an element responsible forbinding to the antigenic epitopes on the groove of the HLA class Imolecules), respectively. CTL clones recognizing the peptide derivedfrom WHSC2 and gene 3, the peptide derived from EIF4EBP1 and gene 4, thepeptide derived from ppMAPkkk and gene 5, expressed TCR possessingdifferent CDR3, respectively. The amino acid sequence of each CDR3 isthat shown with an underline in Table 8-2. TABLE 8-1 CTL clone EpitopesVβ Dβ Jβ Cβ 2-2-H3 UBE2V 43-51 8.1 2.1 2.3 2 2-1-H12 UBE2V 43-51 8.1 2.12.3 2 1-2-D7 HNRPL 140-148 3.2 1.1 2.7 2 1-2-D12 HNRPL 140-148 3.2 1.12.7 2 4-2-A11 WHSC2 103-111 13.1 2.1 2.7 2 4-2-B3 EIF4EBP1 51-59 8.1 1.11.1 1 0.5-1-H2 ppMAPkkk 18 1.1 1.1 1 432-440 1-2-D1 2-5 OAS3 666-674 142.1 2.3 2 2-2-B4 2-5 OAS3 666-674 14 2.1 2.3 2

TABLE 8-2 CTL clone Vβ Dβ* Cβ 2-2-H3 IYFNNNVPIDDSGMPE SLGLAGGEQFFGPGEDLKNVFPPE DRFSAKMPNASFSTLK TRLTVL IQPSEPRDSAVYFCAS 2-1-H12IYFNNNVPIDDSGMPE SLGLAGGEQFFGPG EDLKNVFPPE DRFSAKMPNASFSTLK TRLTVLIQPSEPRDSAVYFCAS 1-2-D7 VSREKKERFSLILESA SLDRSYEQYFGPGT EDLKNVFPPESTNQTSMYLCAS RLTVT 1-2-D12 VSREKKERFSLILESA SLDRSYEQYFGPGT EDLKNVFPPESTNQTSMYLCAS RLTVT 4-2-A11 QGEVPNGYNVSRSTTE SYGGGSSYEQYFGP EDLKNVFPPEDFPLRLLSAAPSQTSV GTRLTVT YFCAS 4-2-B3 IYFNNNVPIDDSGMPE SRVSGEAFFGQGTREDLKNVFPPE DRFSAKMPNASFSTLK LTVV IQPSEPRDSAVYFCAS 0.5-1-H2DESGMPKERFSAEFPK SPTELDTEAFFGQG EDLKNVFPPE EGPSILRIQQVVRGDS TRLTVVAAYFCAS 1-2-D1 VSRKEKRNFPLILESP GGSTDTQYFGPGTR EDLKNVFPPE SPNQTSLYFCASLTVL 2-2-B4 VSRKEKRNFPLILESP GGSTDTQYFGPGTR EDLKNVFPPE SPNQTSLYFCAS LTVL

INDUSTRIAL APPLICABILITY

According to the present invention, HLA-A2-restricted cytotoxic Tlymphocytes can be induced, which makes it possible to achieve aspecific immunotherapy for pancreatic cancer, colon cancer, and stomachcancer. HLA-A2 all eles are found in 23% of African Blacks, 53% ofChinese, 40% of Japanese, 49% of Northern Caucasians, and 38% ofSouthern Caucasians. Consequently, the present invention can be expectedits great contribution to cancer therapy. Moreover, the presentinvention greatly contributes to fundamental research on the moleculerelated to recognition by T cells of a pancreatic cancer cell, a coloncancer cell, stomach cancer, and so on.

1-36. (canceled)
 37. An isolated polypeptide consisting of the aminoacid sequence of SEQ ID NO:48 or a peptide fragment thereof, whereinsaid fragment is selected from the group consisting of: SEQ ID NO:14 andSEQ ID NO:15.
 38. A composition comprising a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:48 or a peptide fragment thereof,wherein said fragment is selected from the group consisting of: SEQ IDNO:14 and SEQ ID NO: 15, and a pharmaceutically acceptable carrier. 39.A method for inducing cytotoxic T lymphocytes, comprising contactinglymphocytes with the composition of claim 38, wherein the lymphocytescontain HLA-A2-restricted cytotoxic T lymphocytes.